HAS2 splicing variant HOEFC11: a target in chronic renal failure, inflammatory diseases and myocardial ischemia

ABSTRACT

HOEFC11 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing HOEFC11 polypeptides and polynucleotides in the design of protocols for the treatment of chronic renal failure, inflammatory diseases, myocardial ischemia, cancer, rheumatoid arthritis, cirrhotic liver disease, among others, and diagnostic assays for such conditions.

This application is a division of application number 08/865,273 filedMay 29, 1997, now U.S. Pat. No. 5,994,100 whose contents areincorporated herein by reference in their entieties.

FIELD OF INVENTION

This invention relates to newly identified polynucleotides, polypeptidesencoded by them and to the use of such polynucleotides and polypeptides,and to their production. More particularly, the polynucleotides andpolypeptides of the present invention relate to the hyaluronan synthasefamily, hereinafter referred to as HOEFC11. The invention also relatesto inhibiting or activating the action of such polynucleotides andpolypeptides.

BACKGROUND OF THE INVENTION

Hyaluronic acid (HA), an important constituent of extracellular matrix,is a linear polysaccharide of alternating glucuronic acid and N-acetylglucosamine residues. It is synthesized by a membrane-bound enzymehyaluronan synthase (HAS) and extruded into the extracellular space.Cloning of two human HAS (HAS 1 and HAS 2) has been reported veryrecently (K. Watanabe and Y. Yamaguchi, J. Biol. Chem. 271:22945-22948,1996) (N. Itano and K. Kimata, Biochem. Biophy. Res. Communications,222:816-820, 1996). HA synthesis is involved in many cellular functionssuch as migration, invasion, adhesion, transformation, proliferation andwound healing. HA synthesis has been shown to be induced by FBS, PDGF,EGF, IL-1, retinoic acid, IGF, TGF beta, etc. Increased HA productionis: (a) a general phenomenon in various organs attacked by inflammatorycells, (b) implicated in tissue edema, (c) a characteristic of tissueremodeling and (d) a marker for early stage of extracellular matrixremodeling following vascular injury. Increased levels of HA have beenreported in chronic renal failure, inflammatory diseases, cancer(prostate, mammary and other invasive tumors), aortas from diabeticpatients, smaller airways of patients with acute alveolitis,transplantation edema in rejecting heart and kidney, myocardialischemia, balloon injury, liver cirrhosis, wound healing andangiogenesis. Hyaluronidase (breaks down HA) is reported to bebeneficial in limiting cellular damage during myocardial ischemia inrat, dog and man. This indicates that the hyaluronan synthase family hasa established, proven history as therapeutic targets. Clearly there is aneed for identification and characterization of further members of thehyaluronan synthase family which can play a role in preventing,ameliorating or correcting dysfunctions or diseases, including, but notlimited to, chronic renal failure, inflammatory diseases, myocardialischemia, cancer, rheumatoid arthritis, cirrhotic liver disease.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to HOEFC11 polypeptides andrecombinant materials and methods for their production. Another aspectof the invention relates to methods for using such HOEFC11 polypeptidesand polynucleotides. Such uses include the treatment of chronic renalfailure, inflammatory diseases, myocardial ischemia, cancer, rheumatoidarthritis, cirrhotic liver disease, among others. In still anotheraspect, the invention relates to methods to identify agonists andantagonists using the materials provided by the invention, and treatingconditions associated with HOEFC11 imbalance with the identifiedcompounds. Yet another aspect of the invention relates to diagnosticassays for detecting diseases associated with inappropriate HOEFC11activity or levels.

DESCRIPTION OF THE INVENTION Definitions

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“HOEFC11” refers, among others, generally to a polypeptide having theamino acid sequence set forth in SEQ ID NO:2 or an allelic variantthereof.

“HOEFC11 activity or HOEFC11 polypeptide activity” or “biologicalactivity of the HOEFC11 or HOEFC11 polypeptide” refers to the metabolicor physiologic function of said HOEFC11 including similar activities orimproved activities or these activities with decreased undesirableside-effects. Also included are antigenic and immonogenic activities ofsaid HOEFC11.

“HOEFC11 gene” refers to a polynucleotide having the nucleotide sequenceset forth in SEQ ID NO:1 or allelic variants thereof and/or theircomplements.

“Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

“Isolated” means altered “by the hand of man” from the natural state. Ifan “isolated” composition or substance occurs in nature, it has beenchanged or removed from its original environment, or both. For example,a polynucleotide or a polypeptide naturally present in a living animalis not “isolated,” but the same polynucleotide or polypeptide separatedfrom the coexisting materials of its natural state is “isolated”, at theterm is employed herein.

“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications has been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

“Polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from posttranslation natural processes ormay be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., “Analysis for protein modifications and nonproteincofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et al., “ProteinSynthesis: Posttranslational Modifications and Aging”, Ann NY Acad Sci(1992) 663:48-62.

“Variant” as the term is used herein, is a polynucleotide or polypeptidethat differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniquesor by direct synthesis.

“Identity” is a measure of the identity of nucleotide sequences or aminoacid sequences. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. See, e.g.:(COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS,Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OFSEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, vonHeinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER,Gribskov, M. and Devereaux, J., eds., M Stockton Press, New York, 1991).While there exist a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCS program package (Devereaux, J., et al., NucleicAcids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F.et al., J Molec Biol (1990) 215:403).

As an illustration, by a polynucleotide having a nucleotide sequencehaving at least, for example, 95% “identity” to a reference nucleotidesequence of SEQ ID NO:1 is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence of SEQ ID NO:1. Inother words, to obtain a polynucleotide having a nucleotide sequence atleast 95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These mutations of the reference sequence may occur at the 5or 3 terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence.

Similarly, by a polypeptide having an amino acid sequence having atleast, for example, 95% “identity” to a reference amino acid sequence ofSEQ ID NO:2 is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of SEQ ID NO:2. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a reference amino acid sequence, up to 5% of the amino acidresidues in the reference sequence may be deleted or substituted withanother amino acid, or a number of amino acids up to 5% of the totalamino acid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

Polypeptides of the Invention

In one aspect, the present invention relates to HOEFC11 polypeptides.The HOEFC11 polypeptides include the polypeptide of SEQ ID NO:2; as wellas polypeptides comprising the amino acid sequence of SEQ ID NO:2; andpolypeptides comprising the amino acid sequence which have at least 80%identity to that of SEQ ID NO:2 over its entire length, and still morepreferably at least 90% identity, and even still more preferably atleast 95% identity to SEQ ID NO:2. Furthermore, those with at least97-99% are highly preferred. Also included within HOEFC11 polypeptidesare polypeptides having the amino acid sequence which have at least 80%identity to the polypeptide having the amino acid sequence of SEQ IDNO:2 over its entire length, and still more preferably at least 90%identity, and still more preferably at least 95% identity to SEQ IDNO:2. Furthermore, those with at least 97-99% are highly preferred.Preferably HOEFC11 polypeptide exhibit at least one biological activityof HOEFC11.

The HOEFC11 polypeptides may be in the form of the “mature” protein ormay be a part of a larger protein such as a fusion protein. It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, pro-sequences, sequences which aid inpurification such as multiple histidine residues, or an additionalsequence for stability during recombinant production.

Fragments of the HOEFC11 polypeptides are also included in theinvention. A fragment is a polypeptide having an amino acid sequencethat entirely is the same as part, but not all, of the amino acidsequence of the aforementioned HOEFC11 polypeptides. As with HOEFC11polypeptides, fragments may be “free-standing,” or comprised within alarger polypeptide of which they form a part or region, most preferablyas a single continuous region. Representative examples of polypeptidefragments of the invention, include, for example, fragments from aboutamino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the endof HOEFC11 polypeptide. In this context “about” includes theparticularly recited ranges larger or smaller by several, 5, 4, 3, 2 or1 amino acid at either extreme or at both extremes.

Preferred fragments include, for example, truncation polypeptides havingthe amino acid sequence of HOEFC11 polypeptides, except for deletion ofa continuous series of residues that includes the amino terminus, or acontinuous series of residues that includes the carboxyl terminus ordeletion of two continuous series of residues, one including the aminoterminus and one including the carboxyl terminus. Also preferred arefragments characterized by structural or functional attributes such asfragments that comprise alpha-helix and alpha-helix forming regions,beta-sheet and beta-sheet-forming regions, turn and turn-formingregions, coil and coil-forming regions, hydrophilic regions, hydrophobicregions, alpha amphipathic regions, beta amphipathic regions, flexibleregions, surface-forming regions, substrate binding region, and highantigenic index regions. Other preferred fragments are biologicallyactive fragments. Biologically active fragments are those that mediateHOEFC11 activity, including those with a similar activity or an improvedactivity, or with a decreased undesirable activity. Also included arethose that are antigenic or immunogenic in an animal, especially in ahuman.

Preferably, all of these polypeptide fragments retain the biologicalactivity of the HOEFC11, including antigenic activity. Variants of thedefined sequence and fragments also form part of the present invention.Preferred variants are those that vary from the referents byconservative amino acid substitutions—i.e., those that substitute aresidue with another of like characteristics. Typical such substitutionsare among Ala, Val, Leu and Ile; among Ser and Thr; among the acidicresidues Asp and Glu; among Asn and Gln; and among the basic residuesLys and Arg; or aromatic residues Phe and Tyr. Particularly preferredare variants in which several, 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination.

The HOEFC11 polypeptides of the invention can be prepared in anysuitable manner. Such polypeptides include isolated naturally occurringpolypeptides, recombinant produced polypeptides, synthetically producedpolypeptides, or polypeptides produced by a combination of thesemethods. Means for preparing such polypeptides are well understood inthe art.

Polynucleotides of the Invention

Another aspect of the invention relates to HOEFC11 polynucleotides.HOEFC11 polynucleotides include isolated polynucleotides which encodethe HOEFC11 polypeptides and fragments, and polynucleotides closelyrelated thereto. More specifically, HOEFC11 polynucleotide of theinvention include a polynucleotide comprising the nucleotide sequenceset fort in SEQ ID NO:1 encoding a HOEFC11 polypeptide of SEQ ID NO:2,and polynucleotide having the particular sequence of SEQ ID NO:1.HOEFC11 polynucleotides further include a polynucleotide comprising anucleotide sequence that has at least 80% identity to a nucleotidesequence encoding the HOEFC11 polypeptide of SEQ ID NO:2 over its entirelength, and a polynucleotide that is at least 80% identical to thathaving SEQ ID NO:1 over its entire length. In this regard,polynucleotides at least 90% identical are particularly preferred, andthose with at least 95% are especially preferred. Furthermore, thosewith at least 97% are highly preferred and those with at least 98-99%are most highly preferred, with at least 99% being the most preferred.Also included under HOEFC11 polynucleotides are a nucleotide sequencewhich has sufficient identity to a nucleotide sequence contained in SEQID NO:1 to hybridize under conditions useable for amplification or foruse as a probe or marker. The invention also provides polynucleotideswhich are complementary to such HOEFC11 polynucleotides.

HOEFC11 of the invention is structurally related to other proteins ofthe hyaluronan synthase family, as shown by the results of sequencingthe cDNA of Table 1 (SEQ ID NO:1) encoding human HOEFC11. The cDNAsequence of SEQ ID NO:1 contains an open reading frame (nucleotidenumber 152 to 974) encoding a polypeptide of 241 amino acids of SEQ IDNO:2. The amino acid sequence of Table 2 (SEQ ID NO:2) has about 99.5%identity (using FASTA) in 210 amino acid residues with hyaluronansynthase (HAS2) (K. Watanabe and Y. Yamaguchi, J. Biol. Chem.271:22945-22948, 1996). Most importantly, HOEFC11 is a naturallyoccurring truncation of the HAS2, missing 342 amino acids at thecarboxyl terminus. The nucleotide sequence of Table 1 (SEQ ID NO:1) hasabout 99.7% identity (using FASTA) in 877 nucleotide residues withhyaluronan synthase (HAS2) (K. Watanabe and Y. Yamaguchi, J. Biol. Chem.271:22945-22948). Most importantly, HOEFC11 is a naturally occurringtruncation of HAS2, missing 1026 bp at the 3′ end of the coding region

TABLE 1^(a) GCACGAGCTGAAGTGCAACGGAAACATAAAGAGAATATTA 40GTGAAATTATTTTTTAAAGTGGGGAAgAATCAAACATTTA 80AgACTCCCCTATCCTTTTTAAATGTTGTTTTTAAATTTCT 120TATTTTTTTTGGCCGGTCGTCTCAAATTCATCTGATCTCT 160TATTACCTCAATTTTGGAAACTGCCCGCCACCGACCCTCC 200GGGACCACACAGACaGGCTGAGGACgACTTTATGACCAAG 240AGCTGAACAAGATGCATTGTGAGAGGTTTCTATGTATCCT 280GAGAATAATTGGAACCACACTCTTTGGAGTCTCTCTCCTC 320CTTGGAATCaCAGCTGCTTATATTGTTGGCTACCAGTTTA 360TCCAAACGGATAATTACTATTTCTCTTTTGGACTGTATGG 400TGCCTTTTTGGCATCACACCTCATCATCCAAAGCCTGTTT 440GCCTTTTTGGAGCACCGAAAAATGAAAAAATCCCTAGAAA 480CCCCCATAAAGTTGAACAAAACAGTTGCCCTTTGCATCGC 520TGCCTATCAAGAAGATCCAGACTACTTAAGGAAATGTTTG 560CAATCTGTGAAAAGGCTAACCTACCCTGGGATTAAAGTTG 600TCATGGTCATAGATGGGAACTCAGAAGATGACCTTTACAT 640GAtGGACATCTTCAGTGAAGTCATGGGCAGAGACAAATCA 680GCCACTCATATcTGGAAGAACAACTTCCACGAAAAGGGTC 720CCGGTGAGACAGATGAGTCACATAAAGAAAGCTCGCAACA 760CGTAACGCAATTGGTCTTGTCCAACAAAAGTATcTGCATC 800ATGCAAAAATGGGGTGGAAAAAGAGAAGTCATGTACACAG 840CCTTCAGAGCACTGGGACGAAGTGTGGATTATGTACAGGT 880AGGTCTCCACATTCCTGCCAGGGCAAACATACATTTAAAT 920AAAGCCGCTTTTGTATCTGTCCAGTCATATGCTATAGCCC 960ATCCTTGTCCCTTCTGAACACAGTACTTCTTTCAGTTCAT 1000TTGAAAACAGCATGACTGTTGAAAGCACATTTTGAAAAAA 1040 AAAAAAAAAAA 1051 ^(a)Anucleotide sequence of a huuan HOEFC11 (SEQ ID NO: 1).

TABLE 2^(b) MHCERFLCILRIIGTTLFGVSLLLGITAAYIVGYQFIQTD 40NYYFSFGLYGAFLASHLIIQSLFAFLEHRKMKKSLETPIK 80LNKTVALCIAAYQEDPDYLRKCLQSVKRLTYPGIKVVMVI 120DGNSEDDLYMMDIFSEVMGRDKSATHIWKNNFHEKGPGET 160DESHKESSQHVTQLVLSNKSICIMQKWGGKREVMYTAFRA 200LGRSVDYVQVGLHIPARANIHLNKAAFVSVQSYAIAHPCP 240 F 241

One polynucleotide of the present invention encoding HOEFC11 may beobtained using standard cloning and screening, from a cDNA libraryderived from mRNA in cells of human osteoblasts using the expressedsequence tag (EST) analysis (Adams, M. D., et al. Science (1991)252:1651-1656; Adams, M. D. et al., Nature, (1992) 355:632-634; Adams,M. D., et al., Nature (1995) 377 Supp:3-174). Polynucleotides of theinvention can also be obtained from natural sources such as genomic DNAlibraries or can be synthesized using well known and commerciallyavailable techniques.

The nucleotide sequence encoding HOEFC11 polypeptide of SEQ ID NO:2 maybe identical to the polypeptide encoding sequence contained in Table 1(nucleotide number 152 to 974 of SEQ ID NO:1)., or it may be a sequence,which as a result of the redundancy (degeneracy) of the genetic code,also encodes the polypeptide of SEQ ID NO:2.

When the polynucleotides of the invention are used for the recombinantproduction of HOEFC11 polypeptide, the polynucleotide may include thecoding sequence for the mature polypeptide or a fragment thereof, byitself; the coding sequence for the mature polypeptide or fragment inreading frame with other coding sequences, such as those encoding aleader or secretory sequence, a pre-, or pro- or prepro- proteinsequence, or other fusion peptide portions. For example, a markersequence which facilitates purification of the fused polypeptide can beencoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., ProcNatl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotidemay also contain non-coding 5′ and 3′ sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

Further preferred embodiments are polynucleotides encoding HOEFC11variants comprise the amino acid sequence HOEFC11 polypeptide of Table 2(SEQ ID NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acidresidues are substituted, deleted or added, in any combination.

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term “stringent conditions” means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences.

Polynucleotides of the invention, which are identical or sufficientlyidentical to a nucleotide sequence contained in SEQ ID NO:1 or afragment thereof, may be used as hybridization probes for cDNA andgenomic DNA, to isolate full-length cDNAs and genomic clones encodingHOEFC11 polypeptide and to isolate cDNA and genomic clones of othergenes that have a high sequence similarity to the HOEFC11 gene. Suchhybridization techniques are known to those of skill in the art.Typically these nucleotide sequences are 80% identical, preferably 95%identical, more preferably 95% identical to that of the referent. Theprobes generally will comprise at least 15 nucleotides. Preferably, suchprobes will have at least 30 nucleotides and may have at least 50nucleotides. Particularly preferred probes will range between 30 and 50nucleotides.

In one embodiment, to obtain a polynucleotide encoding HOEFC11polypeptide comprises the steps of screening an appropriate libraryunder stingent hybridization conditions with a labeled probe having theSEQ ID NO:1 or a fragment thereof; and isolating full-length cDNA andgenomic clones containing said polynucleotide sequence. Thus in anotheraspect, HOEFC11 polynucleotides of the present invention further includea nucleotide sequence comprising a nucleotide sequence that hybridizeunder stringent condition to a nucleotide sequence having SEQ ID NO:1 ora fragment thereof. Also included with HOEFC11 polypeptides arepolypeptide comprising amino acid sequence encoded by nucleotidesequence obtained by the above hybridization condition. Suchhybridization techniques are well known to those of skill in the art.Stringent hybridization conditions are as defined above or alternativelyconditions under overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to animal and human disease.

Vectors, Host Cells, Expression

The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, and hostcells which are genetically engineered with vectors of the invention andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Introduction of polynucleotides into hostcells can be effected by methods described in many standard laboratorymanuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986)and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)such as calcium phosphate transfection, DAEA-dextran mediatedtransfection, transvection, microinjection, cationic lipid-mediatedtransfection, electroporation, transduction, scrape loading, ballisticintroduction or infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used. Such systems include,among others, chromosomal, episomal and virus-derived systems, e.g.,vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression systems may contain control regions that regulate as well asengender expression. Generally, any system or vector suitable tomaintain, propagate or express polynucleotides to produce a polypeptidein a host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those set forth in Sambrook etal., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the desired polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

If the HOEFC11 polypeptide is to be expressed for use in screeningassays, generally, it is preferred that the polypeptide be produced atthe surface of the cell. In this event, the cells may be harvested priorto use in the screening assay. If HOEFC11 polypeptide is secreted intothe medium, the medium can be recovered in order to recover and purifythe polypeptide; if produced intracellularly, the cells must first belysed before the polypeptide is recovered. HOEFC11 polypeptides can berecovered and purified from recombinant cell cultures by well-knownmethods including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatographyis employed for purification. Well known techniques for refoldingproteins may be employed to regenerate active conformation when thepolypeptide is denatured during isolation and or purification.

Diagnostic Assays

This invention also relates to the use of HOEFC11 polynucleotides foruse as diagnostic reagents. Detection of a mutated form of HOEFC11 geneassociated with a dysfunction will provide a diagnostic tool that canadd to or define a diagnosis of a disease or susceptibility to a diseasewhich results from under-expression, over-expression or alteredexpression of HOEFC11. Individuals carrying mutations in the HOEFC11gene may be detected at the DNA level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR or other amplification techniques prior toanalysis. RNA or cDNA may also be used in similar fashion. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to labeled HOEFC11 nucleotide sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase digestion or by differences in melting temperatures.DNA sequence differences may also be detected by alterations inelectrophoretic mobility of DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing. See, e.g., Myers et al.,Science (1985) 230:1242. Sequence changes at specific locations may alsobe revealed by nuclease protection assays, such as RNase and S1protection or the chemical cleavage method. See Cotton et al., Proc NatlAcad Sci USA (1985) 85:4397-4401. In another embodiment, an array ofoligonucleotides probes comprising HOEFC11 nucleotide sequence orfragments thereof can be constructed to conduct efficient screening ofe.g., genetic mutations. Array technology methods are well known andhave general applicability and can be used to address a variety ofquestions in molecular genetics including gene expression, geneticlinkage, and genetic variability. (See for example: M. Chee et al.,Science, Vol 274, pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or determining asusceptibility to chronic renal failure, inflammatory diseases,myocardial ischemia, cancer, rheumatoid arthritis, cirrhotic liverdisease through detection of mutation in the HOEFC11 gene by the methodsdescribed.

In addition, chronic renal failure, inflammatory diseases, myocardialischemia, cancer, rhuematoid arthritis, cirrhotic liver disease, can bediagnosed by methods comprising determining from a sample derived from asubject an abnormally decreased or increased level of HOEFC11polypeptide of HOEFC11 mRNA. Decreased or increased expression can bemeasured at the RNA level using any of the methods, well known in theart for the quantitation of polynucleotides, such as, for example, PCR,RT-PCR, RNase protection, Northern blotting and other hybridizationmethods. Assay techniques that can be used to determine levels of aprotein, such as an HOEFC11 polypeptide, in a sample derived from a hostare well-known to those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis andELISA assays.

Chromosome Assays

The nucleotide sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data. Such data arefound, for example, in V. McKusick, Mendelian Inheritance in Man(available on line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes).

The differences in the cDNA or genomic sequence between affected andunaffected individuals can also be determined. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be the causative agent ofthe disease.

Antibodies

The polypeptides of the invention or their fragments or analogs thereof,or cells expressing them can also be used as immunogens to produceantibodies immunospecific for the HOEFC11 polypeptides. The term“immunospecific” means that the antibodies have substantially greateraffinity for the polypeptides of the invention than their affinity forother related polypeptides in the prior art.

Antibodies generated against the HOEFC11 polypeptides can be obtained byadministering the polypeptides or epitope-bearing fragments, analogs orcells to an animal, preferably a nonhuman, using routine protocols. Forpreparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler, G. and Milstein, C.,Nature (1975) 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today (1983) 4:72) andthe EBV-hybridoma technique (Cole et al., MONOCLONAL ANTIBODIES ANDCANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can also be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms including other mammals, may be used to express humanizedantibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography.

Antibodies against HOEFC11 polypeptides may also be employed to treatchronic renal failure, inflammatory diseases, myocardial ischemia,cancer, rheumatoid arthritis, cirrhotic liver disease, among others.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in a mammal which comprises inoculating themammal with HOEFC11 polypeptide, or a fragment thereof, adequate toproduce antibody and/or T cell immune response to protect said animalfrom chronic renal failure, inflammatory diseases, myocardial ischemia,cancer, rheumatoid arthritis, cirrhotic liver disease, among others. Yetanother aspect of the invention relates to a method of inducingimmunological response in a mammal which comprises, delivering HOEFC11polypeptide via a vector directing expression of HOEFC11 polynucleotidein vivo in order to induce such an immunological response to produceantibody to protect said animal from diseases.

Further aspect of the invention relates to an immunological/vaccineformulation (composition) which, when introduced into a mammalian host,induces an immunological response to that mammal to a HOEFC11polypeptide wherein the composition comprises a HOEFC11 polypeptide orHOEFC11 gene. The vaccine formulations may further comprise a suitablecarrier. Since HOEFC11 polypeptide may be broken down in the stomach, itis preferably administered parenterally (including subcutaneous,intramuscular, intravenous, intradermal etc. injection). Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation instonic with theblood of the recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents or thickening agents. Theformulation may be presented in unit-dose or multi-dose containers, forexample, sealed ampoules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use. The vaccine formulation may also includeadjuvant systems for enhancing the immunogenicity of the formulation,such as oil-in water systems and other systems known in the art. Thedosage will depend on the specific activity of the vaccine and can bereadily determined by routine experimentation.

Screening Assays

The HOEFC11 polypeptide of the present invention may be employed in ascreening process for compounds which activate (agonists) or inhibitactivation of (antagonists, or otherwise called inhibitors) the HOEFC11polypeptide of the present invention. Thus, polypeptides of theinvention may also be used to assess identify agonist or antagonistsfrom, for example, cells, cell-free preparations, chemical libraries,and natural product mixtures. These agonists or antagonists may benatural substrates, ligands, receptors, etc., as the case may be, of thepolypeptide of the present invention; or may be structural or functionalmimetics of the polypeptide of the present invention. See Coligan etal., Current Protocols in Immunology 1(2):Chapter 5 (1991).

HOEFC11 polypeptides are responsible for many biological functions,including many pathologies. Accordingly, it is desirous to findcompounds and drugs which stimulate HOEFC11 polypeptide on the one handand which can inhibit the function of HOEFC11 polypeptide on the otherhand. In general, agonists are employed for therapeutic and prophylacticpurposes for such conditions as chronic renal failure, inflammatorydiseases, myocardial ischemia, cancer, rheumatoid arthritis, cirrhoticliver disease. Antagonists may be employed for a variety of therapeuticand prophylactic purposes for such conditions as chronic renal failure,inflammatory diseases, myocardial ischemia, cancer, rheumatoidarthritis, cirrhotic liver disease.

In general, such screening procedures may involve using appropriatecells which express the HOEFC11 polypeptide or respond to HOEFC11polypeptide of the present invention. Such cells include cells frommammals, yeast, Drosphila or E. coli. Cells which express the HOEFC11polypeptide (or cell membrane containing the expressed polypeptide) orrespond to HOEFC11 polypeptide are then contacted with a test compoundto observe binding, or stimulation or inhibition of a functionalresponse. The ability of the cells which were contacted with thecandidate compounds is compared with the same cells which were notcontacted for HOEFC11 activity.

The assays may simply test binding of a candidate compound whereinadherence to the cells bearing the HOEFC11 polypeptide is detected bymeans of a label directly or indirectly associated with the candidatecompound or in an assay involving competition with a labeled competitor.Further, these assays may test whether the candidate compound results ina signal generated by activation of the HOEFC11 polypeptide, usingdetection systems appropriate to the cells bearing the HOEFC11polypeptide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed.

The HOEFC11 cDNA, protein and antibodies to the protein may also be usedto configure assays for detecting the effect of added compounds on theproduction of HOEFC11 mRNA and protein in cells. For example, an ELISAmay be constructed for measuring secreted or cell associated levels ofHOEFC11 protein using monoclonal and polyclonal antibodies by standardmethods known in the art, and this can be used to discover agents whichmay inhibit or enhance the productions of HOEFC11 (also calledantagonist or agonist, respectively) from suitably manipulated cells ortissues.

The HOEFC11 protein may be used to identify membrane bound or solublereceptors, if any, through standard receptor binding techniques known inthe art. These include, but are not limited to, ligand binding andcrosslinking assays in which the HOEFC11 is labeled with a radioactiveisotope (eg 125I), chemically modified (eg biotinylated), or fused to apeptide sequence suitable for detection or purification, and incubatedwith a source of the putative receptor (cells, cell membranes, cellsupernatants, tissue extracts, bodily fluids). Other methods includebiophysical techniques such as surface plasmon resonance andspectroscopy. In addition to being used for purification and cloning ofthe receptor, these binding assays can be used to identify agonists andantagonists of HOEFC11 which compete with the binding of HOEFC11 to itsreceptors. Standard methods for conducting screening assays are wellunderstood in the art.

Examples of potential HOEFC11 polypeptide antagonists include antibodiesor, in some cases, oligonucleotides or proteins which are closelyrelated to the ligands, substrates, receptors, etc., as the case may be,of the HOEFC11 polypeptide, e.g., a fragment of the ligands, substrates,receptors, or small molecules which bind to the polypeptide of thepresent invention but do not elicit a response, so that the activity ofthe polypeptide is prevented.

Prophylactic and Therapeutic Methods

This invention provides methods of treating abnormal conditions such as,chronic renal failure, inflammatory diseases, myocardial ischemia,cancer, rheumatoid arthritis, cirrhotic liver disease, related to bothan excess of and insufficient amounts of HOEFC11 polypeptide activity.

If the activity of HOEFC11 polypeptide is in excess, several approachesare available. One approach comprises administering to a subject aninhibitor compound (antagonist) as hereinabove described along with apharmaceutically acceptable carrier in an amount effective to inhibitthe function of the HOEFC11 polypeptide, such as, for example, byblocking the binding of ligands, substrates, etc., or by inhibiting asecond signal, and thereby alleviating the abnormal condition. Inanother approach, soluble forms of HOEFC11 polypeptides still capable ofbinding the ligand, substrate, etc. in competition with endogenousHOEFC11 polypeptide may be administered. Typical embodiments of suchcompetitors comprise fragments of the HOEFC11 polypeptide.

In another approach, soluble forms of HOEFC11 polypeptides still capableof binding the ligand in competition with endogenous HOEFC11 polypeptidemay be administered. Typical embodiments of such competitors comprisefragments of the HOEFC11 polypeptide.

In still another approach, expression of the gene encoding endogenousHOEFC11 polypeptide can be inhibited using expression blockingtechniques. Known such techniques involve the use of antisensesequences, either internally generated or separately administered. See,for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Alternatively, oligonucleotides whichform triple helices with the gene can be supplied. See, for example, Leeet al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al., Science (1991) 251:1360. These oligomers can beadministered per se or the relevant oligomers can be expressed ed vivo.

For treating abnormal conditions related to an under-expression ofHOEFC11 and its activity, several approaches are also available. Oneapproach comprises administering to a subject a therapeuticallyeffective amount of a compound which activates HOEFC11 polypeptide,i.e., an agonist as described above, in combination with apharmaceutically acceptable carrier, to thereby alleviate the abnormalcondition. Alternatively, gene therapy may be employed to effect theendogenous production of HOEFC11 by the relevant cells in the subject.For example, a polynucleotide of the invention may be engineered forexpression in a replication defective retroviral vector, as discussedabove. The retroviral expression construct may then be isolated andintroduced into a packaging cell transduced with a retroviral plasmidvector containing RNA encoding a polypeptide of the present inventionsuch that the packaging cell now produces infectious viral particlescontaining the gene of interest. These producer cells may beadministered to a subject for engineering cells in vivo and expressionof the polypeptide in vivo. For overview of gene therapy, see Chapter20, Gene Therapy and other Molecular Genetic-based TherapeuticApproaches, (and references cited therein) in Human Molecular Genetics,T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996). Anotherapproach is to administer a therapeutic amount of HOEFC11 polypeptidesin combination with a suitable pharmaceutical carrier.

Formulation and Administration

Peptides, such as the soluble form of HOEFC11 polypeptides, and agonistsand antagonist peptides or small molecules, may be formulated incombination with a suitable pharmaceutical carrier. Such formulationscomprise a therapeutically effective amount of the polypeptide orcompound, and a pharmaceutically acceptable carrier or excipient. Suchcarriers include but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof.Formulation should suit the mode of administration, and is well withinthe skill of the art. The invention further relates to pharmaceuticalpacks and kits comprising one or more containers filled with one or moreof the ingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

Preferred forms of systemic administration of the pharmaceuticalcompositions include injection, typically by intravenous injection.Other injections routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if properly formulated in enteric or encapsulatedformulations, oral administration may also be possible. Administrationof these compounds may also be topical and/or localized, in the form ofsalves, pastes, gels and the like.

The dosage range required depends on the choice of peptide, the route ofadministration, the nature of the formulation, the nature of thesubject's condition, and the judgement of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.Wide variations in the needed dosage, however, are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in thesubject, in treatment modalities often referred to as “gene therapy” asdescribed above. Thus, for example, cells from a subject may beengineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples illustrate, but do not limitthe invention.

Example 1

HAS 2 has 6 predicted potential transmembrane domains, 2 in theN-terminal and 4 in the C-terminal regions (K. Watanabe and Y.Yamaguchi, J. Biol. Chem. 271:22945-22948, 1996). In the middle of thepolypeptide, there are 5 amino acid residues that are thought to becrucial for the N-acetylglucosaminyltransferase activity in theStreptococcus HA synthase (S. Nagahashi, et al., J. Biol. Chem.270:13961-13967, 1995). The synthesis of HA increases in proliferatingfibroblasts while it is inhibited in growth-arrested cells (M. Brecht,et al., Biochem. J. 239:445-450, 1986; K. Matuoka, et al., J. cell Biol.104:1105-1115, 1987; J. R. Kitchen, et al., Biolchem. J. 309:649-656,1995). However, little is known about the regulation of HA synthesis.Here, we identified a novel splicing variant of HAS2, HOEFC11, whichmissed the 5 crucial amino acids for the enzyme activity and the 4transmembrane domains in the C-terminus. This variant form of HAS2 mayplay a regulatory role in the HA synthesis by acting as a dominantnegative inhibitor of HAS2 enzyme. This mechanism has been welldemonstrated in the study of aldehyde dehydrogenase, ornithinetranscarboxylase, as well as many membrane-bound receptors (Y. Nakamuraand H. Nakauchi, Sci. 264:588-589: R. Ebner, et al., Sci. 260:1344-1348;S. Werner, et al., EMBO. J. 12:1635-2643).

A search of a random cDNA sequence database from Human Genome Sciencesconsisting of short sequences known as expressed sequence tags (ESTs)using BLAST algorithm disclosed an EST (#1750866) which was homologousto human hyaluronan synthase (HAS2). HGS EST 1750866 has the followingsequence:

1 CTGAAGTGCA AGNAAACATA AAGAGAATAT TAGTGAAATT ATTTTTTAAA (SEQ ID NO:3)51 GTGGGGAAGA ATCAAACATT TAAGACTCCC CTATCCTTTT TAAATGTTGT 101 TTTTAAATTTCTTATTTTTT TTGGCCGGTC GTCTCAAATT CATCTGATCT 151 CTTATTACCT CAATTTTGGAAACTGCCCGC CACCGACCCT CCGGGGACCA 201 CACAGACAGG CTGAGGACGA CTTTATGACCAAGAGCTGAA CAAGAGNCAT 251 TGTGAGAGGT TCCAAGGAAC CNGNAGATAA TTGGGANCCAAACCTTTGGN 301 GGT

In order to obtain the full length clones, a complete DNA sequence ofthe inserts were deduced using automated DNA sequencing procedure. Oneof the clones, HOEFC11, contained a 1 kb insert. A map analysis of theDNA sequence using the Lasergene software indicated an open readingframe (ORF) which was a truncated form of HAS2. In order to confirm theidentify of the clone, PCR primers were designed using the nucleotidesequence of the open reading frame (ORF). A DNA fragment with thecorrect size was amplified from human prostate and placenta mRNA andsubcloned into pCR2.1 vector from Invitrogen (San Diego, Calif.). TheDNA sequence was identical to the open reading frame (ORF) of HOEFC11.

3 1051 base pairs nucleic acid single linear cDNA 1 GCACGAGCTGAAGTGCAACG GAAACATAAA GAGAATATTA GTGAAATTAT TTTTTAAAGT 60 GGGGAAGAATCAAACATTTA AGACTCCCCT ATCCTTTTTA AATGTTGTTT TTAAATTTC 120 TATTTTTTTTGGCCGGTCGT CTCAAATTCA TCTGATCTCT TATTACCTCA ATTTTGGAA 180 CTGCCCGCCACCGACCCTCC GGGACCACAC AGACAGGCTG AGGACGACTT TATGACCAA 240 AGCTGAACAAGATGCATTGT GAGAGGTTTC TATGTATCCT GAGAATAATT GGAACCACA 300 TCTTTGGAGTCTCTCTCCTC CTTGGAATCA CAGCTGCTTA TATTGTTGGC TACCAGTTT 360 TCCAAACGGATAATTACTAT TTCTCTTTTG GACTGTATGG TGCCTTTTTG GCATCACAC 420 TCATCATCCAAAGCCTGTTT GCCTTTTTGG AGCACCGAAA AATGAAAAAA TCCCTAGAA 480 CCCCCATAAAGTTGAACAAA ACAGTTGCCC TTTGCATCGC TGCCTATCAA GAAGATCCA 540 ACTACTTAAGGAAATGTTTG CAATCTGTGA AAAGGCTAAC CTACCCTGGG ATTAAAGTT 600 TCATGGTCATAGATGGGAAC TCAGAAGATG ACCTTTACAT GATGGACATC TTCAGTGAA 660 TCATGGGCAGAGACAAATCA GCCACTCATA TCTGGAAGAA CAACTTCCAC GAAAAGGGT 720 CCGGTGAGACAGATGAGTCA CATAAAGAAA GCTCGCAACA CGTAACGCAA TTGGTCTTG 780 CCAACAAAAGTATCTGCATC ATGCAAAAAT GGGGTGGAAA AAGAGAAGTC ATGTACACA 840 CCTTCAGAGCACTGGGACGA AGTGTGGATT ATGTACAGGT AGGTCTCCAC ATTCCTGCC 900 GGGCAAACATACATTTAAAT AAAGCCGCTT TTGTATCTGT CCAGTCATAT GCTATAGCC 960 ATCCTTGTCCCTTCTGAACA CAGTACTTCT TTCAGTTCAT TTGAAAACAG CATGACTG 1020 GAAAGCACATTTTGAAAAAA AAAAAAAAAA A 1051 241 amino acids amino acid single linearprotein 2 Met His Cys Glu Arg Phe Leu Cys Ile Leu Arg Ile Ile Gly ThrThr 1 5 10 15 Leu Phe Gly Val Ser Leu Leu Leu Gly Ile Thr Ala Ala TyrIle Val 20 25 30 Gly Tyr Gln Phe Ile Gln Thr Asp Asn Tyr Tyr Phe Ser PheGly Leu 35 40 45 Tyr Gly Ala Phe Leu Ala Ser His Leu Ile Ile Gln Ser LeuPhe Ala 50 55 60 Phe Leu Glu His Arg Lys Met Lys Lys Ser Leu Glu Thr ProIle Lys 65 70 75 80 Leu Asn Lys Thr Val Ala Leu Cys Ile Ala Ala Tyr GlnGlu Asp Pro 85 90 95 Asp Tyr Leu Arg Lys Cys Leu Gln Ser Val Lys Arg LeuThr Tyr Pro 100 105 110 Gly Ile Lys Val Val Met Val Ile Asp Gly Asn SerGlu Asp Asp Leu 115 120 125 Tyr Met Met Asp Ile Phe Ser Glu Val Met GlyArg Asp Lys Ser Ala 130 135 140 Thr His Ile Trp Lys Asn Asn Phe His GluLys Gly Pro Gly Glu Thr 145 150 155 160 Asp Glu Ser His Lys Glu Ser SerGln His Val Thr Gln Leu Val Leu 165 170 175 Ser Asn Lys Ser Ile Cys IleMet Gln Lys Trp Gly Gly Lys Arg Glu 180 185 190 Val Met Tyr Thr Ala PheArg Ala Leu Gly Arg Ser Val Asp Tyr Val 195 200 205 Gln Val Gly Leu HisIle Pro Ala Arg Ala Asn Ile His Leu Asn Lys 210 215 220 Ala Ala Phe ValSer Val Gln Ser Tyr Ala Ile Ala His Pro Cys Pro 225 230 235 240 Phe 303base pairs nucleic acid single linear cDNA 3 CTGAAGTGCA AGNAAACATAAAGAGAATAT TAGTGAAATT ATTTTTTAAA GTGGGGAAGA 60 ATCAAACATT TAAGACTCCCCTATCCTTTT TAAATGTTGT TTTTAAATTT CTTATTTTT 120 TTGGCCGGTC GTCTCAAATTCATCTGATCT CTTATTACCT CAATTTTGGA AACTGCCCG 180 CACCGACCCT CCGGGGACCACACAGACAGG CTGAGGACGA CTTTATGACC AAGAGCTGA 240 CAAGAGNCAT TGTGAGAGGTTCCAAGGAAC CNGNAGATAA TTGGGANCCA AACCTTTGG 300 GGT 303

What is claimed is:
 1. An isolated polypeptide comprising the amino acidsequence of SEQ ID NO:2.
 2. The isolated polypeptide of claim 1consisting of the amino acid sequence of SEQ ID NO:2.